Using Accelerator Mass Spectrometry to

Explain the Pharmacokinetics of

VismodegibCornelis E.C.A. Hop

Topics to be Addressed

• Why AMS?

• AMS for mass balance studies with

vismodegib

• AMS for absolute bioavailability studies with

vismodegib

Vismodegib - Approved by the FDA for the

Treatment of BCC in January 2012

MW = 421

pKa = 2.8

clogP = 3.9

Human PK Predictions

• Predicted human PK:• Low clearance: < 1 ml/min/kg

• Lower volume of distribution: < 1 ml/min/kg

• Long half life

• Incomplete absorption:• Rat F = 53%; Dog F = 33 %

• Based on BDC rat and dog data, Fa is 42% and 31%, respectively

F = Fa * Fg * Fh

• Minimal turnover in microsomes

and hepatocytes for all species

except cyno; Human Clhep = 0.5

mL/min/kg

• Low clearance in rat and dog,

but moderate in cyno

Clinical PK of VismodegibPhase I Cancer Patient Study

A B

C (A) Increase in exposure after single 150 or

270 mg dose with sustained [plasma]

(B) Steady state reached quickly;

unexpected with 10-14 day half-life

(C) Steady state concentration independent

of dose

(D) Particle size effect on day 1 that was

not present at steady state (data not

shown)

Graham et al., Clin Cancer Res 2011, 17, 2512

5

EOP1 FDA Comments

• According to 21 CFR 320.25, the bioavailability (fraction

absorbed by IV/PO routes) of GDC-0449 should be

assessed.

• Absolute bioavailability study

• Address the absorption, distribution, metabolism and

excretion (ADME study) of GDC-0449 in humans and if

needed characterize the effect of renal and or hepatic

impairment on the PK of GDC-0449.

• Mass balance study

… But How?

• Traditional mass balance study:• How ethical is it to have healthy volunteers exposed

to 50 – 100 mCi for several months?• Can you keep them in the clinic that long?

• The amount excreted each day is likely very small and may not be detectable with liquid scintillation counting

• Poor mass balance?

• Can metabolites in plasma, feces and urine be detected?

Mass Balance Studies for Compounds with Long T1/2

• Poor mass balance is likely for vismodegibRoffey et al., Drug Metab Rev 2007, 39, 17

vismodegib

… But How?

• Traditional mass balance study:• How ethical is it to have healthy volunteers exposed

to 50 – 100 mCi for several months?• Can you keep them in the clinic that long?

• The amount excreted each day is likely very small and may not be detectable with liquid scintillation counting

• Poor mass balance

• Can metabolites in plasma, feces and urine be detected?

• Study is likely to fail

• 14C tracer study with accelerator mass spectrometry detection!

AMS allows quantitative separation of 12C, 13C

and 14C from a sample containing a very low

level 14C-tagged drug or biopharmaceutical

Accelerator Mass Spectrometer

Flow Diagram of AMS Procedures

for Biomedical Samples

Analyse by AMS

Administer nCi 14C-drug Collect blood etc

CombustGraphitise

Types of AMS Studies in Drug Development

Phase 0

Human micro-dosing study Drug candidate selection using human micro-dosing data

Investment decisions

Phase I/II

IV PK, Human Absolute Bioavailability study Intravenous tracer + concomitant oral dose

Mass balance and nano-tracing human metabolism study Low dose of 14C incorporated into the therapeutic oral dose of drug

Mass balance and metabolite profiling – As an alternative to conventional Human Radiolabel Study?

– Addresses MIST guidelines

To answer specific ADME questions

To investigate/mitigate metabolism liabilities & retention issues

Mass balance and metabolite profiling

Microtracer Absolute Bioavailability Studies with

NCEs Intended for Regulatory Submission

13

Courtesy of Xceleron & Graham Lappin

AMS Mass Balance Study Design

• Amount of radioactivity greatly reduced: 1,000 nCi

• It is feasible to send volunteers home after two

weeks

Time (days)

Cp/D

ose

150 mg PO on day 1 (unlabeled)

+ 1,000 nCi/10 µg 14C PO

Cold

Hot

How to ensure acceptable mass

balance?

Continuous sampling until day 14

and interim sampling on days 21,

28, 35, 42, 49, and 56 and

interpolation

Dose as homogenous suspension

Vismodegib Mass Balance Results

Total Recovery = 86.6%Confined D1-D14

Feces Recovery

= 82.2%

Urine Recovery = 4.4%

• Renal impairment study not needed - widens exclusion criteria for ongoing trials

• Need for hepatic impairment study dependent on metabolite profiling

Vismodegib Predominant in Plasma

Time (hr)

0 200 400 600 800 1000 1200 1400

Pla

sm

a C

once

ntr

atio

n (

ng o

r n

g e

qu

iv p

er

mL

)

0.1

1

10

100

1000

10000

Plasma GDC-0449 "cold" (ng/mL)

Plasma total radioactivity "hot" (ng equiv/mL)

GDC-0449 is major drug-species in plasma - no significant circulating metabolites

Graham et al., Drug Metab Dispos 2011, 39, 1460

Metabolic Profiling in Feces

• Mainly vismodegib in circulation

• Mainly excreted in feces as parent compound

(including un-absorbed material) and oxidative

metabolites

GDC-0449

M3dp

m/g

fec

es h

om

og

en

ate

GDC-0449

M3dp

m/g

fec

es h

om

og

en

ate

feces 0-72 hrs

GDC-0449

M3

M1

M13

M18

feces 72-312 hrs

Graham et al., Drug Metab Dispos 2011, 39, 1460

Metabolism of Vismodegib

Oxidative metabolism

and unique pyridine ring

opening

Graham et al., Drug Metab Dispos 2011, 39, 1460

AMS Absolute Bioavailability Study Design

• Dose cold vismodegib orally and 14C labeled

vismodegib intravenously at the Tmax

• No concerns about linearity of iv PK because hot and

cold present at the same time

• Single dose and steady state (one week dosing)

Time (days)

Cp/d

ose

150 mg PO

on day 1

500 nCi/10 µg 14C IV dose

on day 1 at Tmax = 2 hr

Time (days)

Cp/d

ose

150 mg PO qd

for 7 days

500 nCi/10 µg 14C

IV dose on day 7

Vismodegib Absolute Bioavailability ResultsDose Normalized GDC-0449 Concentration vs. Time

Time (hours)

0 200 400 600 800 1000 1200 1400

Dose n

orm

alized G

DC

-0449 P

lasm

a C

oncentr

ation

(ng/m

L/d

ose)

0.01

0.1

1

10

100

1000

Oral Mean

IV Mean

Dose Normalized GDC-0449 Concentration vs. Time

Time (hours)

0 200 400 600 800 1000 1200 1400

Dose n

orm

alized G

DC

-0449 P

lasm

a C

oncentr

ation

(ng/m

L/d

ose)

0.01

0.1

1

10

100

1000

Oral Mean

IV Mean

• Slow IV CL consistent with rate limited systemic elimination (not “flip-flop”)

• Absolute bioavailability determined to be 31.8 + 4.6%

• Does F and/or CL change with daily dosing?

• Total clearance increased close to two-fold

• Absolute bioavailability decreased to 7.4 + 2.5%

dose-normalized

data

Graham et al., Br J Clin.Pharmacol 2012, 73, 788

T1/2

(d)

CL

(mL/h)

Vss

(L)

Single

dose13.0 43.4 16.4

Multiple

dose10.3 78.5 26.8

Summary of Vismodegib IV PK

0.01 mL/min/kg

+81%

No evidence for CYP induction in human hepatocytes

Graham et al., Br J Clin.Pharmacol 2012, 73, 788

Plasma Protein Binding

• Binding to both HSA and AAG, but AAG binding can be saturated• The higher concentration at steady state results in a larger free

fraction

Graham et al., Br J Clin.Pharmacol 2012, 73, 788

T1/2

(d)

CL

(mL/h)

CLu

(mL/h)

Vss

(L)

Vss,u

(L)

Single

dose13.0 43.4 13,152 16.4 4,970

Multiple

dose10.3 78.5 9,937 26.8 3,392

Summary of Vismodegib IV PK

• The intrinsic clearance and volume of distribution does

not change significantly from single to multiple dose

Graham et al., Br J Clin.Pharmacol 2012, 73, 788

T1/2

(d)

CL

(mL/h)

CLu

(mL/h)

Vss

(L)

Vss,u

(L)

Oral

AUC

(mM.hr)

F

(%)

Single

dose13.0 43.4 13,152 16.4 4,970 2,850 31.8

Multiple

dose10.3 78.5 9,937 26.8 3,392 356 7.4

Summary of Vismodegib IV & Oral PK

• The total oral exposure is reduced by close to eight-fold

due to non-sink absorption conditions

-88% -77%

Graham et al., Br J Clin.Pharmacol 2012, 73, 788

Summary of Vismodegib Human

Pharmacokinetics• Vismodegib is absorbed slowly after a single oral dose mainly due to

poor solubility

• Terminal elimination half-life is very long after both oral and IV administration due to very slow elimination

• The CL is incredibly low, 0.01 mL/min/kg

• Metabolism is via oxidation and pyridine ring opening

• The Vss of 0.23 L/kg is low, but is still indicative of distribution out of the plasma space.

• Mean bioavailability is moderate, 31.8%, with little inter-individual variability; micromolar plasma concentrations were achieved with a single 150 mg oral dose

• At steady state, the absorption and clearance are reduced, but the intrinsic clearance has not changed

• All of this is driven by very slow clearance, non-sink absorption and non-linear plasma protein binding

• AMS enabled these studies and absolute bioavailability and mass balance studies using AMS are now a standard part of drug development

We Can Rationalize the Clinical PK of Vismodegib!Phase I Cancer Patient Study

A B

C (A) Increase in exposure after single 150 or

270 mg dose with sustained [plasma]

(B) Steady state reached quickly;

unexpected with 10-14 day half-life

(C) Steady state concentration independent

of dose

(D) Particle size effect on day 1 that was

not present at steady state (data not

shown)

Graham et al., Clin Cancer Res 2011, 17, 2512

Acknowledgements

• DMPK, Clinical

Pharmacology and

Clinical Operations

colleagues and others

on the HH team

• PRA and Xceleron and

all healthy volunteers

History of Accelerator Mass Spectrometry

• Highly specialised nuclear physics instrument

originally developed for carbon dating in the 1970’s

• It uses very high energies to separate rare isotopes

which are then measured with high precision

• Willard Frank Libby won the Nobel Prize for Chemistry

for his method to use carbon-14 for age determination

in archaeology, geology, geophysics

• Approximately 160 AMS instruments worldwide

• First biological application described in 1989 (LLNL)

• Xceleron first company in the world to use AMS for

biomedical research

AMS

Atoms separated by differences 12C,13C and 14C atoms

individually countedin mass, charge and energy

-decay of 14C atom Detected by LSC as photons

of light in photomultiplier tube

LSC

0.012% of 14C decays per annum; 1 billion 14C atoms ≡ 1 dpm

Key

Sample containing 12C 13C and 14C atoms

Principle of LSC vs AMS Detection

1000 14C atoms required for valid measurement

1,000,000 more sensitive than LSC

100,000 more sensitive than MS

Xceleron’s 5 MV AMS

0.01

0.1

1

0 500 1000 1500 2000 2500

Time (h)

Lo

g p

erc

en

t o

f to

tal

ex

cre

ted

pe

r 2

4 h

pe

rio

d

Up to 168 h all

excreta collected

After 168 h, collections over

selected 24 h periods

AUC0- = 90.5 % recovery

Subjects were administered 3 mg/1 μCi of drug X orally

Interpolation to Ensure Adequate Radioactivity

Recovery for Long Half-Life Drugs